High-throughput label-free enzymatic bioassays using DESI-MS

ABSTRACT

The invention generally relates to high-throughput label-free enzymatic bioassays using desorption electrospray ionization-mass spectrometry (DESI-MS).

RELATED APPLICATION

The present application is a continuation of U.S. nonprovisionalapplication Ser. No. 17/193,120, filed Mar. 5, 2021, which claims thebenefit of and priority to U.S. provisional patent application Ser. No.63/022,715, filed May 11, 2020, the content of each of which is hereinincorporated by reference in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under W911NF-16-2-0020awarded by the Army Research Office on behalf of the Defense AdvancedProjects Research Agency. The government has certain rights in theinvention.

FIELD OF THE INVENTION

The invention generally relates to high-throughput label-free enzymaticbioassays using desorption electrospray ionization-mass spectrometry(DESI-MS).

BACKGROUND

Enzymes are important drug targets. Many marketed drugs today functionthrough inhibition of enzymes mediating disease phenotypes. Enzymeinhibitors are an important class of pharmacological agents. Often thesemolecules are competitive, reversible inhibitors of substrate binding.

To that end, enzyme assays are important tools for measuring cellularactivity and for monitoring enzyme proteins. Measurement of enzymekinetics provides crucial information on the mechanisms of enzymecatalysis and on the interactions of enzymes with substrates,inhibitors, drugs, and drug candidates.

SUMMARY

The invention provides a high-throughput platform that combines a set ofinterrelated methods in which mass spectrometry methods may be used tocreate droplets and thin films in which reactions occur (optionallyaccelerated reactions in certain embodiments), while simultaneously orsubsequently using mass spectrometry to analyze the product distributionin the droplets and/or thin films. This platform has broad applicabilityto many different chemical and biological systems. In embodimentsdescribed herein, this platform is applied to enzymatic reactions andenzymatic bioassays.

In certain aspects, the invention provides methods for monitoring anenzymatic reaction. These methods involve preparing a plurality ofdiscrete spots on a substrate. In certain embodiments, each of theplurality of discrete spots is from a different time point in anenzymatic reaction. In other embodiments, each of the discrete spots maybe from the same time point for different reactions with differentinhibitor/reactivator/substrate/enzyme concentrations. For example, aset up based on a same end point would be more common for drugdiscovery, whereas a set-up based on different time points would becommon for biochemical studies and enzyme characterization.

Each of the discrete spots may comprise a substrate and a product(s) ofthe enzymatic reaction. The methods then involve directing sequentiallya discharge from an ionization source onto each of the plurality ofdiscrete spots to sequentially desorb the substrate and/or product(s)from each of the discrete spots and sequentially generate ions of thesubstrate and/or product(s) from each of the discrete spots that enter amass spectrometer, and analyzing sequentially the ions of the substrateand/or product(s) from each of the discrete spots in the massspectrometer to thereby monitor the enzymatic reaction. As mentionedabove, in certain embodiments, each of the plurality of discrete spotsmay be from a different time point in an enzymatic reaction that hasbeen stopped prior to the preparation step. In other embodiments, eachof the plurality of discrete spots is from a different time point in anenzymatic reaction that has not been stopped prior to the preparationstep. In certain embodiments a compound is added to the reaction mixtureto serve as an internal standard for quantitation.

In other aspects, the invention provides methods for monitoring anenzymatic reaction that involve preparing a plurality of discrete spotson a substrate. In certain embodiments a compound is added to thereaction mixture to serve as an internal standard for quantitation. Eachof the discrete spots may comprise a substrate and a product of theenzymatic reaction. Such methods additionally involve directingsequentially a discharge from an ionization source onto each of theplurality of discrete spots to sequentially desorb the substrate and/orproduct(s) from each of the discrete spots and sequentially generateions of the substrate and/or product(s) from each of the discrete spotsthat enter a mass spectrometer, obtaining sequentially ion intensitiesof the ions of the substrate and/or product(s) from each of the discretespots in the mass spectrometer, and converting ion intensities of theions of the substrate and/or product(s) from each of the discrete spotsinto concentration ratios of the substrate and product(s) by applying acalibration curve, thereby monitoring the enzymatic reaction. In certainembodiments, each of the plurality of discrete spots is from a differenttime point in an enzymatic reaction that has been stopped prior to thepreparation step. In other embodiments, each of the plurality ofdiscrete spots is from a different time point in an enzymatic reactionthat has not been stopped prior to the preparation step. In certainembodiments, the methods measure not necessarily the ratio of productand substrate but the ratio of either of those and an internal standard.

In certain embodiments of the above methods, the ionization source is adesorption electrospray ionization (DESI) probe and the discharge is aDESI spray.

In certain embodiments of the above methods, a subset of each of theplurality of the discrete spots further comprises a same test compound.In such embodiments, the method further comprises determining whetherthe test compound inhibits the enzymatic reaction based on themonitoring of the progress of the enzymatic reaction. In suchembodiments, the method further comprises determining how completely thetest compound inhibits the enzymatic reaction based on the monitoring ofthe progress of the enzymatic reaction. In such embodiments, theenzymatic reaction is associated with a physiological condition anddetermining how completely the test compound inhibits the enzymaticreaction determines whether the test compound should be considered fordevelopment into a drug to treat the condition.

In other embodiments, a subset of each of the plurality of the discretespots further comprises an inhibitor of an enzyme of the enzymaticreaction and a test compound. In such embodiments, the method furthercomprises determining whether the test compound can counteract theinhibitor and re-start the enzymatic reaction. In such embodiments, themethod further comprises determining how completely the test compoundcounteracts the inhibitor and how completely the enzymatic reaction isre-started based on the monitoring of the progress of the enzymaticreaction. In such embodiments, determining how completely the testcompound counteracts the inhibitor and how completely the enzymaticreaction is re-started determines whether the test compound should beconsidered for development into a drug that counteracts the inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 describes an overview of the high-throughput platform.

FIG. 2 describes another embodiment of the high-throughput platform.

FIGS. 3A-D describe an exemplary work-flow using an embodiment of thehigh-throughput platform.

FIG. 4 . is a graph showing representative examples of the spectraobtained for the monitoring of enzymatic reactions using the platformdescribed herein directly from the bioassay mixture containing bothsurfactants and non-volatile buffers.

FIG. 5 shows that empirical progress of the reaction calculated from theDESI-MS data (using intensities of the ions (m/z 104)/(m/z 146+m/z 104))can be directly converted into actual reaction progress([choline]/([acetylcholine]+[choline]) using a simple calibration curvebetween the measured ion ratios and the actual concentration ratios ofcholine and acetylcholine in the mixture.

FIG. 6 shows that progress of the enzymatic reaction can be readilyobtained by quenching aliquots of the bioassay mixture every 1-5 minutesand measuring choline ion ratio (using intensities of the ions (m/z104)/(m/z 146+m/z 104)) vs. time.

FIG. 7 shows that the Michaelis-Menten plot for acetylcholinesterase(AChE) was built using 18 independent reaction progress curves(independent triplicates of six substrate concentrations: 0.4, 0.5, 0.6,0.7, 0.8, 1.0 mM).

FIG. 8 shows that using higher concentrations of enzyme and substrate, 9μg/mL and 5 mM, respectively, the reaction can be completed in fiveminutes and 384 inhibition reactions are analyzed in <15 min whileexcellent sensitivity is achieved.

FIG. 9 shows that robustness of the approach was checked using adifferent DESI source and a different mass spectrometer (Waters sourceand Synapt G2 qToF).

FIG. 10 panels A-D show that inhibitors of interest (i.e. which showrelatively high activity compared to a known inhibitor in the initialhigh-low screening) can be then further studied.

FIG. 11 shows that further characterization of the inhibitors wasachieved by screening simultaneously different concentrations ofinhibitor and the substrate in order to obtain the Dixon plot from whichthe inhibition constant was rapidly determined as 70.0±0.7 nM.

FIG. 12 panels A-C show that acetylcholinesterase reactivators can bescreened against different inhibitors of interest, characterizing thedynamics of both inhibition and reactivation.

FIG. 13 panel A shows a simulated inhibition plot used to assess theperformance of the approach. FIG. 13 panel B shows that thelog-transformed data shows the expected linear behavior (R2=0.996).

FIG. 14 panels A-B show representative mass spectra of a bioassaymixture (panel A) just after enzyme addition and (panel B) after 30minutes of room temperature incubation (in 0.1 M phosphate buffer, pH 8,0.1% BSA).

FIG. 15 panel A shows simulated log-inhibition plot used to assess theperformance of the approach using a different mass spectrometer (WatersQToF). FIG. 15 panel B shows a typical spectrum obtained with ourapproach using the QToF instrument.

FIG. 16 is an illustration showing an exemplary data analysis module forimplementing the systems and methods of the invention in certainembodiments.

DETAILED DESCRIPTION

The invention generally relates to high-throughput label-free enzymaticbioassays using desorption electrospray ionization-mass spectrometry(DESI-MS). FIG. 1 describes an overview of the high-throughput platform.The fluid handling system controls sample handling and generatesmicrowell plates of various sizes, exemplified here as a 384 well plate.The fluid handling system using a pinning device that interacts with themicrowell plate to generate a substrate of discrete spots, using forexample the pinning device. A DESI source is integrated as part of thesystem and directs a DESI active spray discharge onto each of the spotssequentially. The spray discharge desorbs and ionizes analytes from eachspot, which are directed into a mass spectrometer for analysis. Thishigh-throughput system has the capability of screening reaction productsof interest at 1 second per reaction (or less) and is a system capableof 24-hour continuous operation. FIG. 2 describes another embodiment ofthe high-throughput platform with certain additional features. FIGS.3A-D describe an exemplary work-flow using an embodiment of thehigh-throughput platform.

DESI is an ambient ionization method that allows the direct ionizationof species from a sample, and is described in each of Takats et al.,Science, 306:471-473, 2004 and Takats, U.S. Pat. No. 7,335,897, thecontent of each of which is incorporated by reference herein in itsentirety. DESI-MS imaging is described for example in Eberlin et al.(Biochimica Et Biophysica Acta 2011, 1811(11):946-960) and Cooks R G, etal. (Faraday Discussions 2011, 149:247-267), the content of each ofwhich is incorporated by reference herein in its entirety.

As mentioned above, this platform has broad applicability to manydifferent chemical and biological systems. For example, the set ofcapabilities includes the ability, based on the measured mass spectra,to (i) read the contents of a microarray at high speed using DESI and tocapture this information and process it automatically, (ii) reread thecontents of a microarray at high speed using DESI after subjecting thesamples in the array to exposure to chemical reagents or biologicalagents, (iii) use DESI to react mixtures of compounds present in themicroarray, and simultaneously, record mass spectra of the secondaryDESI droplets to characterize the reaction products, (iv) use DESI withadded reagent in the spray (reactive DESI) to react (derivatize)compounds in the array and simultaneously to record mass spectra of thesecondary droplets to characterize the derivatized compounds, and (v)use DESI to characterize biomarkers and other features of tissue inarray format. In some cases, the DESI analysis conditions might bechosen to minimize the possibility of accelerated reaction in asecondary microdroplets. In some cases, the sample to MS inlet and otheranalysis parameters might be chosen to maximize the possibility of suchaccelerated reactions occurring.

The information obtained from reading the contents of a microarray athigh speed using DESI allows for (i) optimization of synthesisconditions for maximum yields and/or product purities when the reactionsstudied are scaled up using flow chemistry or conventional bulksynthesis, (ii) systematic studies of reaction mechanisms over a rangeof conditions by characterization of reaction intermediates as anadditional route to information that will facilitate reactions on anyscale, (iii) collection of small amounts of synthetic product from thedroplet reactions on a receiver surface, (iii) measurement of thecytotoxicity of the small amounts of material collected by fluorescencemeasurements as is well known in the state of the art, (iv)characterization of the purity of the collected product by independentMS and/or MS/MS measurements, (v) compilation of the optical and MS/MSspectra on the small amounts of collected new compounds in a databasefor forensic and other uses, (vi) measurement of binding assays by labelfree MS after exposing the collected product to an enzyme or receptor orother substrate, and (vii) determination of structure/activityrelationships using bioactivity measurements on collected products.Certain embodiments focus on measurement of binding assays by label freeMS after exposing the collected product to an enzyme or receptor orother substrate.

The platform described herein was used to perform enzymatic bioassays ina high-throughput and label-free manner (FIG. 4 ). Initial efforts werefocused on studying the acetylcholinesterase (AChE) assay, which isrelevant in the context of drug discovery for Alzheimer's disease aswell as the development of contra-measures against chemical warfareagents. To develop the bioassays the substrate (acetylcholine, m/z 146)and product (choline, m/z 104) of the enzymatic reaction weresimultaneously monitored. The analysis was performed using DESI-MS(spray solvent: MeOH-ACN 1:1) directly from the bioassay mixture(enzyme, phosphate buffer 0.1 M pH 8, 0.1% BSA) after quenching with ACN(up to 50% final concentration) and pinning (50 nL) on PTFE-coated glassslides. The effective analysis time per sample (spot) is as little as0.3 seconds, in such a way that a 384-sample array can be analyzedcompletely in 7 minutes. Kinetic studies are readily performed byquenching aliquots after fixed time intervals (typically 1-2 minutes).The figure illustrates the quality of the spectra obtained over the m/zrange of interest, as well as the behavior of the product and substratethroughout the reaction. The reaction time to completion can beoptimized by selecting appropriate amounts of enzyme and substrate,depending on the objective of the study.

FIG. 5 shows that empirical progress of the reaction calculated from theDESI-MS data (using intensities of the ions (m/z 104)/(m/z 146+m/z 104))can be directly converted into actual reaction progress([choline]/([acetylcholine]+[choline]) using a simple calibration curvebetween the measure ion ratios and the actual concentration ratios ofcholine and acetylcholine in the mixture. The calibration curve shownhere was built using 6 independent solutions of different totalconcentrations (range: 0.4-1.0 mM), that underwent the same procedure asdid the bioassay samples (ACN addition and pinning). Note that theamount of material used for the analysis is on the order of 100-300 pg.Each data point represents the average of the six solutions, eachanalyzed four times on three different days, for a total of 72measurements per concentration ratio. The acquisition time for thisdaily calibration (246 spots) is around 5 minutes. This calibrationallows direct determination of kinetic parameters of the enzymaticreaction without the need for labeled compounds (either deuteratedstandards or non-native substrates).

FIG. 6 shows progress of the enzymatic reaction can be readily obtainedby quenching aliquots of the bioassay mixture every 1-5 minutes andmeasuring choline ion ratio relative to acetylcholine+choline vs. time.For kinetic determinations the reactions were incubated for 50 minutes,with the concentration of substrate held kept between 0.4 to 1 mM, whilethe concentration of enzyme was set to 180 ng/mL. The linear section ofthe curve was converted to choline concentration, using the calibrationcurve shown in FIG. 5 . Note that only concentrations within thecalibration range were used. The calibrated result (inset) is used todetermine v0 of the reaction. The data shown come from eightinstrumental replicates from a single mixture (128 spots), acquired inless than 2.5 minutes.

FIG. 7 shows that the Michaelis-Menten plot for AChE was built using 18independent reaction progress curves (independent triplicates of sixsubstrate concentrations: 0.4, 0.5, 0.6, 0.7, 0.8, 1.0 mM). The curvewas used to estimate the Michaelis-Menten constant of the system as0.203±0.006 mM, which is within the range of values reported in theliterature (0.08 to 0.23 mM). Construction of this plot, by measuringeight instrumental replicates of each data point in each reactionprogress curve, required the analysis of 2304 spots, which can bereadily done in around 30 minutes. Note that the incubation (50 minutes)for all the samples can be performed simultaneously, so that only 80minutes in total is required for the kinetic characterization of thereaction.

FIG. 8 shows that using higher concentrations of enzyme and substrate, 9μg/mL and 5 mM, respectively, the reaction can be completed in fiveminutes and excellent sensitivity is achieved. Under these conditions 34compounds were tested as potential inhibitors of AChE: 1 known stronginhibitor (neostigmine), 17 natural products and 16 drugs of abuse. Thecompounds were tested at two different concentrations (high and low: 0.6and 0.06 mM), and both positive and negative controls were analyzed inthe same assay. All the samples were analyzed in quadruplicate (for atotal of 280 spots), the whole set requiring less than 6 minutes ofanalysis time. Note that if no replicates are analyzed, 384 inhibitorscan be all tested in less than 15 minutes total (5-minuteincubation+7-minute analysis+2-minute data processing).

Robustness of the approach was checked using a different DESI source anda different mass spectrometer (Waters source and Synapt G2 qToF) (FIG. 9). Results for the inhibition assays are almost identical to those shownin the heatmap of FIG. 8 (Prosolia source and Thermo LTQ ion trap).

Inhibitors of interest (i.e. which show relatively high activitycompared to a known inhibitor in the initial high-low screening) can bethen further studied (FIGS. 10A-D). From the original set of 34compounds screened four—neostigmine, oxycodone, hydrocodone andcaffeine—were selected and dose-response curves were obtained for all ofthem. With these curves the IC50 values of these compounds under ourparticular assay conditions were found as 5.9, 0.122, 116 and 71 μM forneostigmine, oxycodone, hydrocodone and caffeine, respectively, whichagrees with the expected trend. Each of these dose-response curvesrepresents the average of three independent solutions with eightinstrumental replicates each. In total, 1536 reactions were analyzed toobtain the four curves presented here, the total analysis requiringaround 25 minutes.

Further characterization of the inhibitors was achieved by screeningsimultaneously different concentrations of inhibitor and the substratein order to obtain the Dixon plot (FIG. 11 ). Neostigmine was chosen forthis further characterization. This Dixon plot required that 24independent progress curves be determined: duplicate measurements takenon 12 combinations of concentrations of inhibitor (0, 40, 100, 200 nM)and substrate (0.6, 0.8, 1.0 mM). This required the analysis of 3072spots, carried out in approx. 35 minutes, with simultaneous incubationtime of 50 minutes. As expected, the position of the intersection of thelines in the plot indicates that neostigmine is a competitive inhibitor.Its inhibition constant was determined as 70.0±0.7 nM, a valuecomparable to those reported for this enzyme-inhibitor complex. FIG. 12shows that the dynamics of enzymatic inhibition-reactivation processescan be studied. The figure shows the inhibition of acetylcholinesterasethroughout its incubation with 5 μM dichlorovos (panel A), 0.25 μMchlorpyrifos-oxon (panel B), and 0.75 μM paraoxon (panel C), as well asthe enzyme reactivation due to the addition of a commercial reactivator(2-pralidoxime, 100 μM). Addition of the oxime reactivator is indicatedby the solid gray line in the plots. Reactivation efficiencies of 45±2%,87±5% and 76±3% were observed after inhibition with dichlorovos,chlorpyrifos-oxon, and paraoxon, respectively.

System Architecture

In certain embodiments, the systems and methods of the invention can becarried out using automated systems and computing devices. Specifically,aspects of the invention described herein can be performed using anytype of computing device, such as a computer, that includes a processor,e.g., a central processing unit, or any combination of computing deviceswhere each device performs at least part of the process or method. Insome embodiments, systems and methods described herein may be controlledusing a handheld device, e.g., a smart tablet, or a smart phone, or aspecialty device produced for the system.

Systems and methods of the invention can be performed using software,hardware, firmware, hardwiring, or combinations of any of these.Features implementing functions can also be physically located atvarious positions, including being distributed such that portions offunctions are implemented at different physical locations (e.g., imagingapparatus in one room and host workstation in another, or in separatebuildings, for example, with wireless or wired connections).

Processors suitable for the execution of computer program include, byway of example, both general and special purpose microprocessors, andany one or more processor of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read-only memory ora random access memory or both. The essential elements of computer are aprocessor for executing instructions and one or more memory devices forstoring instructions and data. Generally, a computer will also include,or be operatively coupled to receive data from or transfer data to, orboth, one or more mass storage devices for storing data, e.g., magnetic,magneto-optical disks, or optical disks. Information carriers suitablefor embodying computer program instructions and data include all formsof non-volatile memory, including by way of example semiconductor memorydevices, (e.g., EPROM, EEPROM, solid state drive (SSD), and flash memorydevices); magnetic disks, (e.g., internal hard disks or removabledisks); magneto-optical disks; and optical disks (e.g., CD and DVDdisks). The processor and the memory can be supplemented by, orincorporated in, special purpose logic circuitry.

To provide for interaction with a user, the subject matter describedherein can be implemented on a computer having an I/O device, e.g., aCRT, LCD, LED, or projection device for displaying information to theuser and an input or output device such as a keyboard and a pointingdevice, (e.g., a mouse or a trackball), by which the user can provideinput to the computer. Other kinds of devices can be used to provide forinteraction with a user as well. For example, feedback provided to theuser can be any form of sensory feedback, (e.g., visual feedback,auditory feedback, or tactile feedback), and input from the user can bereceived in any form, including acoustic, speech, or tactile input.

The subject matter described herein can be implemented in a computingsystem that includes a back-end component (e.g., a data server), amiddleware component (e.g., an application server), or a front-endcomponent (e.g., a client computer having a graphical user interface ora web browser through which a user can interact with an implementationof the subject matter described herein), or any combination of suchback-end, middleware, and front-end components. The components of thesystem can be interconnected through network by any form or medium ofdigital data communication, e.g., a communication network. For example,the reference set of data may be stored at a remote location and thecomputer communicates across a network to access the reference set tocompare data derived from the female subject to the reference set. Inother embodiments, however, the reference set is stored locally withinthe computer and the computer accesses the reference set within the CPUto compare subject data to the reference set. Examples of communicationnetworks include cell network (e.g., 3G or 4G), a local area network(LAN), and a wide area network (WAN), e.g., the Internet.

The subject matter described herein can be implemented as one or morecomputer program products, such as one or more computer programstangibly embodied in an information carrier (e.g., in a non-transitorycomputer-readable medium) for execution by, or to control the operationof, data processing apparatus (e.g., a programmable processor, acomputer, or multiple computers). A computer program (also known as aprogram, software, software application, app, macro, or code) can bewritten in any form of programming language, including compiled orinterpreted languages (e.g., C, C++, Perl), and it can be deployed inany form, including as a stand-alone program or as a module, component,subroutine, or other unit suitable for use in a computing environment.Systems and methods of the invention can include instructions written inany suitable programming language known in the art, including, withoutlimitation, C, C++, Perl, Java, ActiveX, HTMLS, Visual Basic, orJavaScript.

A computer program does not necessarily correspond to a file. A programcan be stored in a file or a portion of file that holds other programsor data, in a single file dedicated to the program in question, or inmultiple coordinated files (e.g., files that store one or more modules,sub-programs, or portions of code). A computer program can be deployedto be executed on one computer or on multiple computers at one site ordistributed across multiple sites and interconnected by a communicationnetwork.

A file can be a digital file, for example, stored on a hard drive, SSD,CD, or other tangible, non-transitory medium. A file can be sent fromone device to another over a network (e.g., as packets being sent from aserver to a client, for example, through a Network Interface Card,modem, wireless card, or similar).

Writing a file according to the invention involves transforming atangible, non-transitory computer-readable medium, for example, byadding, removing, or rearranging particles (e.g., with a net charge ordipole moment into patterns of magnetization by read/write heads), thepatterns then representing new collocations of information aboutobjective physical phenomena desired by, and useful to, the user. Insome embodiments, writing involves a physical transformation of materialin tangible, non-transitory computer readable media (e.g., with certainoptical properties so that optical read/write devices can then read thenew and useful collocation of information, e.g., burning a CD-ROM). Insome embodiments, writing a file includes transforming a physical flashmemory apparatus such as NAND flash memory device and storinginformation by transforming physical elements in an array of memorycells made from floating-gate transistors. Methods of writing a file arewell-known in the art and, for example, can be invoked manually orautomatically by a program or by a save command from software or a writecommand from a programming language.

Suitable computing devices typically include mass memory, at least onegraphical user interface, at least one display device, and typicallyinclude communication between devices. The mass memory illustrates atype of computer-readable media, namely computer storage media. Computerstorage media may include volatile, nonvolatile, removable, andnon-removable media implemented in any method or technology for storageof information, such as computer readable instructions, data structures,program modules, or other data. Examples of computer storage mediainclude RAM, ROM, EEPROM, flash memory, or other memory technology,CD-ROM, digital versatile disks (DVD) or other optical storage, magneticcassettes, magnetic tape, magnetic disk storage or other magneticstorage devices, Radiofrequency Identification tags or chips, or anyother medium which can be used to store the desired information andwhich can be accessed by a computing device.

As one skilled in the art would recognize as necessary or best-suitedfor performance of the methods of the invention, a computer system ormachines of the invention include one or more processors (e.g., acentral processing unit (CPU) a graphics processing unit (GPU) or both),a main memory and a static memory, which communicate with each other viaa bus.

In an exemplary embodiment shown in FIG. 16 , system 200 can include acomputer 249 (e.g., laptop, desktop, or tablet). The computer 249 may beconfigured to communicate across a network 209. Computer 249 includesone or more processor 259 and memory 263 as well as an input/outputmechanism 254. Where methods of the invention employ a client/serverarchitecture, steps of methods of the invention may be performed usingserver 213, which includes one or more of processor 221 and memory 229,capable of obtaining data, instructions, etc., or providing results viainterface module 225 or providing results as a file 217. Server 213 maybe engaged over network 209 through computer 249 or terminal 267, orserver 213 may be directly connected to terminal 267, including one ormore processor 275 and memory 279, as well as input/output mechanism271.

System 200 or machines according to the invention may further include,for any of I/O 249, 237, or 271 a video display unit (e.g., a liquidcrystal display (LCD) or a cathode ray tube (CRT)). Computer systems ormachines according to the invention can also include an alphanumericinput device (e.g., a keyboard), a cursor control device (e.g., amouse), a disk drive unit, a signal generation device (e.g., a speaker),a touchscreen, an accelerometer, a microphone, a cellular radiofrequency antenna, and a network interface device, which can be, forexample, a network interface card (NIC), Wi-Fi card, or cellular modem.

Memory 263, 279, or 229 according to the invention can include amachine-readable medium on which is stored one or more sets ofinstructions (e.g., software) embodying any one or more of themethodologies or functions described herein. The software may alsoreside, completely or at least partially, within the main memory and/orwithin the processor during execution thereof by the computer system,the main memory and the processor also constituting machine-readablemedia. The software may further be transmitted or received over anetwork via the network interface device.

Multiplexing and Inductive Charging

In certain embodiments, multiplexing on sample loading and analysis isused in the systems and methods of the invention, optionally usinginductive charging for analysis. Such approaches are described forexample in U.S. patent application publication number 2020/0381238, thecontent of which is incorporated by reference herein in its entirety. Incertain embodiments, the induced DC nESI ionization source includes a 3Delectrical controlled moving platform, emitter holder and a pogo pinholder. In such embodiments, the emitter holder is preloaded with 96emitters and samples. The emitter holder is attached to the 3D movingstage by a 3D printed connector. The emitter holder is designed toeasily attached and detached from the moving stage for convenience ofsample introduction and cleaning. The front (side facing the MS inlet)of the emitter holder has 96 holes to hold 96 emitters. Inside theholes, there are 96 individual electrodes with the same length as theemitter holder. When loading the emitters into the holder, theseelectrodes are inserted into the emitters but do not reach the samplesolution. The other ends of the electrodes go from the rear (sideopposite from the MS inlet) and are soldered to a PCB with 96 holes. Onthe PCB, there are 96 isolated copper layers electrically in contactwith the 96 electrodes by soldering. A pogo pin electrode placed behindthe PCB is aligned with the MS inlet. The position of the pogo pinelectrode is fixed by the pogo pin holder on a fixed arm of the 3Dmoving stage. The pogo pin electrode touches the PCB. When the device isrunning, the motion control system first goes to the top right startingpoint and moves in the vertical y-direction to find the first row ofemitters and then moves in the horizontal x-direction to analyze samplesin the first row in sequence. When an emitter is aligned with the MSinlet, the pogo pin touches the corresponding copper layer on the PCBand 2˜3.5 kV volts is applied to the electrode for induced DC nESIionization of the sample in the tip of the emitter. Note that theelectrode does not contact the sample so ionization is induced. Becausethe flow rate in inductive nESI is very low, so there is enough time torecord the high-quality MS data in spite of very small sample volume.

To solve the problem of sample introduction presented by the traditionalnESI work flow, we have developed a “dip and go” strategy using amultiplexed system. In such an approach, 96 emitters with 20-micron tipsize are preloaded into the emitter holder. The size of the holder isdesigned to correspond to the size of the standard 96-well plate and theposition of each emitter corresponds to the position of each well in the96-well plate. To load the sample, one holds the emitter holder and letsthe side with emitters face the 96-well plate, lowers the holder andallows every emitter to be immersed into sample solution for 10 secondsand then lifts the holder. This procedure can be done manually or with arobot. The amount of sample solution introduced into emitter is ca. 100nL. Sample loading amounts can be varied by using different loadingtimes.

Induced electrophoretic cleaning (“desalting”) can be applied to thesamples on the emitters prior to sample analysis to achieve betteranalytical performance for samples with a complex matrix. By applyingvoltage (e.g., more than 5 kV, with either the same or opposite polarityto that used for nESI analysis) to the electrodes simultaneously, thehigh electrical field induced in the sample in the emitter tip willcause electrophoresis. Ions with large ionic mobility such as anions andcations from simple salts in the solution will migrate towards the twoends of the solution, leaving substances with small ionic mobility suchas peptides will remain essentially in their original positions and willbe subject to selective ionization.

To perform offline electrophoretic cleaning one holds the emitter holderand allows the copper layer of the PCB touch a copper plate connected tothe high voltage output of a power supply. At 0.5 to 1 cm distance fromthe emitter tip, another copper plate which is grounded is placed so asto set up a large potential change in the sample solution to initiateelectrophoresis. The electrophoresis is maintained for 10 seconds andthen the emitter holder is re-installed onto the back to the 3D movingstage platform. Following the same steps described in section A onerecords spectra of the cleaned samples. This method is more convenientbut slightly slower (because cleaning slightly slows the rate of motionused for ionization).

The alternative to offline cleaning is to perform online cleaning usingone HV supply for cleaning and a second one for ionization. To performonline electrophoretic cleaning, the emitter holder is attached to themoving stage. When performing the cleaning, the moving stage allows theemitter holder to move from left to right. The left pogo pin on a pogopin holder is supplied with −6 kV volts to induce electrophoreticcleaning of the sample that points towards the grounded counterelectrode. Subsequently, after cleaning, the emitter moves and isaligned with the MS inlet at which point the right pogo pin electrodewith 2 to 3.5 kV volts applied to the pogo pin holder initiatesinductive nESI analysis of sample in the emitter by the same processdescribed in A. This method is faster and the sample screening rate canbe maximized.

As mentioned above, inductive charging can be useful in a multiplexanalysis setting. Inductive charging is further described for example inU.S. Pat. No. 9,184,036, the content of which is incorporated byreference herein in its entirety. In inductive charging the probeincludes a spray emitter and a voltage source and the probe isconfigured such that the voltage source is not in contact with the sprayemitter or the spray emitted by the spray emitter. In this manner, theions are generated by inductive charging, i.e., an inductive method isused to charge the primary microdroplets. This allows droplet creationto be synchronized with the opening of the sample introduction system(and also with the pulsing of the nebulizing gas). Inductive nESI can beimplemented for various kinds of nESI arrays due to the lack of physicalcontact. Examples include circular and linear modes. In an exemplaryrotating array, an electrode placed mm from each of the spray emittersin turn is supplied with a 2-4 kV positive pulse (10-3000 Hz) giving asequence of ion signals. Simultaneous or sequential ions signals can begenerated in the linear array using voltages generated inductively inadjacent nESI emitters. Nanoelectrospray spray plumes can be observedand analytes are detected in the mass spectrum, in both positive andnegative detection modes. In the electrophoretic clean-up working mode,direct current voltage source (1.5-6 kV) was used to inducenanoelectrospray. Different from the previous example induced byalternating current voltage, the induced electrical field keeps the samedirection in this mode, which ensures efficient electrophoretic cleaningperformance.

Ion Traps and Mass Spectrometers

Any ion trap known in the art can be used in systems of the invention.Exemplary ion traps include a hyperbolic ion trap (e.g., U.S. Pat. No.5,644,131, the content of which is incorporated by reference herein inits entirety), a cylindrical ion trap (e.g., Bonner et al.,International Journal of Mass Spectrometry and Ion Physics,24(3):255-269, 1977, the content of which is incorporated by referenceherein in its entirety), a linear ion trap (Hagar, Rapid Communicationsin Mass Spectrometry, 16(6):512-526, 2002, the content of which isincorporated by reference herein in its entirety), and a rectilinear iontrap (U.S. Pat. No. 6,838,666, the content of which is incorporated byreference herein in its entirety).

Any mass spectrometer (e.g., bench-top mass spectrometer of miniaturemass spectrometer) may be used in systems of the invention and incertain embodiments the mass spectrometer is a miniature massspectrometer. An exemplary miniature mass spectrometer is described, forexample in Gao et al. (Anal. Chem. 2008, 80, 7198-7205.), the content ofwhich is incorporated by reference herein in its entirety. In comparisonwith the pumping system used for lab-scale instruments with thousands ofwatts of power, miniature mass spectrometers generally have smallerpumping systems, such as a 18 W pumping system with only a 5 L/min (0.3m3/hr) diaphragm pump and a 11 L/s turbo pump for the system describedin Gao et al. Other exemplary miniature mass spectrometers are describedfor example in Gao et al. (Anal. Chem., 2008, 80, 7198-7205.), Hou etal. (Anal. Chem., 2011, 83, 1857-1861.), and Sokol et al. (Int. J. MassSpectrom., 2011, 306, 187-195), the content of each of which isincorporated herein by reference in its entirety.

The control system of the Mini 12 (Linfan Li, Tsung-Chi Chen, Yue Ren,Paul I. Hendricks, R. Graham Cooks and Zheng Ouyang “Miniature AmbientMass Analysis System” Anal. Chem. 2014, 86 2909-2916, DOI:10.1021/ac403766c; and 860. Paul I. Hendricks, Jon K. Dalgleish, JacobT. Shelley, Matthew A. Kirleis, Matthew T. McNicholas, Linfan Li,Tsung-Chi Chen, Chien-Hsun Chen, Jason S. Duncan, Frank Boudreau, RobertJ. Noll, John P. Denton, Timothy A. Roach, Zheng Ouyang, and R. GrahamCooks “Autonomous in-situ analysis and real-time chemical detectionusing a backpack miniature mass spectrometer: concept, instrumentationdevelopment, and performance” Anal. Chem., 2014, 86 2900-2908 DOI:10.1021/ac403765x, the content of each of which is incorporated byreference herein in its entirety), and the vacuum system of the Mini 10(Liang Gao, Qingyu Song, Garth E. Patterson, R. Graham Cooks and ZhengOuyang, “Handheld Rectilinear Ion Trap Mass Spectrometer”, Anal. Chem.,78 (2006) 5994-6002 DOI: 10.1021/ac061144k, the content of which isincorporated by reference herein in its entirety) may be combined toproduce the miniature mass spectrometer shown in FIG. 9 . It may have asize similar to that of a shoebox (H20 cm×W25 cm×D35 cm). In certainembodiments, the miniature mass spectrometer uses a dual LITconfiguration, which is described for example in Owen et al. (U.S.patent application Ser. No. 14/345,672), and Ouyang et al. (U.S. patentapplication Ser. No. 61/865,377), the content of each of which isincorporated by reference herein in its entirety.

EXAMPLES Example 1

A calibration curve of choline (m/z 104) was obtained by the highthroughput approach and using acetylcholine (0.25 mM, m/z 146) asinternal standard (FIG. 12 ). All samples were prepared in the bioassaymatrix (0.1 M phosphate buffer, pH 8, 0.1% BSA) and analyzed without anypretreatment. Nine individual samples were prepared at eachconcentration (and blank) using the fluidic handling robot, and thenpinned four times as 50 nL spots in different locations on the samePTFE-coated plate, giving a total of 216 spots. DESI-MS was used for theanalysis of the plate in positive ion mode (spray solvent: MeOH, appliedvoltage: 4 kV, instrument: ion trap). Data acquisition and processingwas controlled by the CHRIS software. The effective analysis time of allthe spots was under four minutes. Each data point in the calibrationcurve represents the average of 36 spots (9 individual solutions, 4replicates each). The coefficient of determination (R2) of the linearmodel adjusted to fit the data is 0.998. Results are reproducible afterpinning different slides on different days.

Example 2

FIG. 13 panel A shows a simulated inhibition plot used to assess theperformance of the approach. Eight different ratios ofcholine/acetylcholine (m/z 104/146) were explored, keeping constant thetotal concentration of substrate plus product([choline]+[acetylcholine]=500 μM). These solutions simulated theresults from an inhibition experiment (in which the transformation ofacetylcholine to choline would reduce the acetylcholine ratio as theconcentration of inhibitor increases). The expected behavior (over atleast a region of the curve) is logarithmic. As observed FIG. 13 panelB, the log-transformed data shows the expected linear behavior(R2=0.996). This demonstrates how our approach could be used tocalculate IC50 values without the need for internal standards, labels orsample pretreatment. All solutions were prepared in the bioassay matrix(0.1 M phosphate buffer, pH 8, 0.1% BSA) using the fluidic handlingrobot and then rapidly pinned four times on the same PTFE-coated plate.DESI-MS was used for the analysis of the plate in positive ion mode(spray solvent: MeOH, applied voltage: 4 kV, instrument: Thermo linearion trap). Data acquisition and processing was controlled by the CHRISsoftware. In both plots each data point represents the average of 12spots (3 independent solutions, 4 replicates each). The effectiveanalysis time of all the spots (including blanks) was under 3 minutes.

Example 3

FIG. 14 panels A-B show representative mass spectra of a bioassaymixture (panel A) just after enzyme addition and (panel B) after 30minutes of room temperature incubation (in 0.1 M phosphate buffer, pH 8,0.1% BSA). As it can be observed, acetylcholine (m/z 146) was completelytransformed to choline (m/z 104) after incubation under the experimentalconditions. Data were acquired using DESI-MS in positive ion mode (spraysolvent: MeOH, applied voltage: 4 kV, instrument: ion trap) without anysample pretreatment, just after spotting of the bioassay mixture.

Example 4

FIG. 15 panel A shows simulated log-inhibition plot used to assess theperformance of the approach using a different mass spectrometer (WatersQToF). Data were acquired in a similar fashion to FIG. 14 panels A-B.Three plots (with statistically identical slopes) were obtained by usingthree different ratios of enzyme to substrate. These ratios werecalculated using the intensity of choline (m/z 104) divided by eitherthe molecular ion of acetylcholine (m/z 146), its fragment (m/z 87) orthe sum of both (m/z 146+87). Results behave as expected. FIG. 15 panelB shows a typical spectrum obtained with our approach using the QToFinstrument.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein.

What is claimed is:
 1. A method for analyzing one or more enzymaticreactions, the method comprising: preparing a plurality of discretespots on a substrate, wherein each of the discrete spots comprises asubstrate and a product of one or more enzymatic reactions, wherein bothof the substrate and the product of the one or more enzymatic reactionsare label-free, and wherein a subset of each of the plurality of thediscrete spots further comprises a same test compound; directingsequentially a discharge from an ionization source onto each of theplurality of discrete spots to sequentially desorb the substrate and/orproduct from each of the discrete spots and sequentially generate ionsof the substrate and/or product from each of the discrete spots thatenter a mass spectrometer; analyzing sequentially the ions of thesubstrate and/or product from each of the discrete spots in the massspectrometer to thereby analyzing the one or more enzymatic reactions;and determining whether the test compound inhibits the enzymaticreaction based on monitoring of the progress of the enzymatic reaction.2. The method of claim 1, wherein the ionization source is a desorptionelectrospray ionization probe (DESI) and the discharge is a DESI spray.3. The method of claim 1, further comprising determining how completelythe test compound inhibits the enzymatic reaction based on themonitoring of the progress of the enzymatic reaction.
 4. The method ofclaim 3, wherein the enzymatic reaction is associated with aphysiological condition and determining how completely the test compoundinhibits the enzymatic reaction determines whether the test compoundshould be considered for development into a drug to treat the condition.5. A method for analyzing one or more enzymatic reactions, the methodcomprising: preparing a plurality of discrete spots on a substrate,wherein each of the discrete spots comprises a substrate and a productof one or more enzymatic reactions, wherein both of the substrate andthe product of the one or more enzymatic reactions are label-free andwherein a subset of each of the plurality of the discrete spots furthercomprises an inhibitor of an enzyme of the enzymatic reaction and a testcompound; directing sequentially a discharge from an ionization sourceonto each of the plurality of discrete spots to sequentially desorb thesubstrate and/or product from each of the discrete spots andsequentially generate ions of the substrate and/or product from each ofthe discrete spots that enter a mass spectrometer; analyzingsequentially the ions of the substrate and/or product from each of thediscrete spots in the mass spectrometer to thereby analyzing the one ormore enzymatic reactions; and determining whether the test compound cancounteract the inhibitor and re-start the enzymatic reaction.
 6. Themethod of claim 5, further comprising determining how completely thetest compound counteracts the inhibitor and how completely the enzymaticreaction re-started based on the monitoring of the progress of theenzymatic reaction.
 7. The method of claim 6, wherein determining howcompletely the test compound counteracts the inhibitor and howcompletely the enzymatic reaction is re-started determines whether thetest compound should be considered for development into a drug thatcounteracts the inhibitor.
 8. The method of claim 1, wherein each of theplurality of discrete spots is from a different time point in anenzymatic reaction.
 9. A method for analyzing one or more enzymaticreactions, the method comprising: preparing a plurality of discretespots on a substrate, wherein each of the discrete spots comprises asubstrate and a product of one or more enzymatic reactions, wherein bothof the substrate and the product of the one or more enzymatic reactionsare label-free; directing sequentially a discharge from an ionizationsource onto each of the plurality of discrete spots to sequentiallydesorb the substrate and/or product from each of the discrete spots andsequentially generate ions of the substrate and/or product from each ofthe discrete spots that enter a mass spectrometer; obtainingsequentially ion intensities of the ions of the substrate and/or productfrom each of the discrete spots in the mass spectrometer; and convertingion intensities of the ions of the substrate and/or product from each ofthe discrete spots into concentration ratios of the substrate andproduct by applying a calibration curve, thereby analyzing the one ormore enzymatic reactions.
 10. The method of claim 9, wherein theionization source is a desorption electrospray ionization probe (DESI)and the discharge is a DESI spray.
 11. The method of claim 9, wherein asubset of each of the plurality of the discrete spots further comprisesa same test compound.
 12. The method of claim 9, wherein the methodfurther comprises determining whether the test compound inhibits theenzymatic reaction based on the monitoring of the progress of theenzymatic reaction.
 13. The method of claim 12, further comprisingdetermining how completely the test compound inhibits the enzymaticreaction based on the monitoring of the progress of the enzymaticreaction.
 14. The method of claim 13, wherein the enzymatic reaction isassociated with a condition and determining how completely the testcompound inhibits the enzymatic reaction determines whether the testcompound should be considered for development into a drug to treat thecondition.
 15. The method of claim 9, wherein a subset of each of theplurality of the discrete spots further comprises an inhibitor of anenzyme of the enzymatic reaction and a test compound.
 16. The method ofclaim 15, wherein the method further comprises determining whether thetest compound can counteract the inhibitor and re-start the enzymaticreaction.
 17. The method of claim 16, further comprising determining howcompletely the test compound counteracts the inhibitor and howcompletely the enzymatic reaction re-started based on the monitoring ofthe progress of the enzymatic reaction.
 18. The method of claim 17,wherein determining how completely the test compound counteracts theinhibitor and how completely the enzymatic reaction is re-starteddetermines whether the test compound should be considered fordevelopment into a drug that counteracts the inhibitor.
 19. The methodof claim 17, wherein each of the plurality of discrete spots is from adifferent time point in an enzymatic reaction.